We designed self-excising plasmid vectors. These vectors can be useful in projects requiring switchable yeast genome modifications.

Proteins can aggregate in response to stresses, including hyperosmotic shock. Formation and disassembly of aggregates is a relatively slow process. We describe a novel instant response of the cell to hyperosmosis, during which chaperones and other proteins form numerous foci with properties uncharacteristic of classical aggregates. These foci appeared/disappeared seconds after shock onset/removal, in close correlation with cell volume changes. Genome-wide and targeted testing revealed chaperones, metabolic enzymes, P-body components and amyloidogenic proteins in the foci. Most of these proteins can form large assemblies and for some, the assembled state was pre-requisite for participation in foci. A genome-wide screen failed to identify genes whose absence prevented foci participation by Hsp70. Shapes of and interconnections between foci revealed by super-resolution microscopy indicated that the foci were compressed between other entities. Based on our findings, we suggest a new model of cytosol architecture as a collection of numerous of gel-like regions suspended in a liquid network. This network is reduced in volume in response to hyperosmosis and forms small pockets between the gel-like regions.

The vacuolar Ca2+ ATPase Pmc1 is involved in maintenance of a low Ca2+ concentration in cytosol in yeast cells. Here we observed that increase of Ca2+ cytosolic concentration in yeast Hansenula polymorpha due to inactivation of Pmc1 resulted in sensitivity to sodium dodecyl sulfate (SDS). To elucidate the mechanisms of the observed effect, a screening for mutations suppressing SDS sensitivity of the H. polymorpha pmc1 mutant was performed. As a result, three genes were identified. Two of them, designated as their Saccharomyces cerevisiae orthologs CCH1 and HOG1 encoded the plasma membrane voltage-gated high-affinity calcium channel and the MAP kinase involved in osmoregulation, respectively. The third gene, designated as WEE1, coded for the ortholog of Wee1/Swe1 kinase involved in cell cycle regulation by inhibiting of the G2/M transition. Detailed analysis of this mutant demonstrated that suppression of pmc1 SDS sensitivity by the wee1 mutation depended on an accompanying chromosomal rearrangement, whereas inactivation of WEE1 in the absence of this rearrangement caused SDS sensitivity. Expression of a chimeric protein containing an N-terminal portion of Wee1 in the pmc1 mutant led to abnormal morphology characteristic of G2 delay. Our data indicate that cytosolic Ca2+ rise causes SDS sensitivity in H. polymorpha through the activation of the Wee1 kinase, which is mediated by the Hog1 kinase. Wee1 has a dual role in the manifestation of SDS sensitivity in the H. polymorpha pmc1 mutant. Mechanisms of influence of the obtained mutations on the G2/M transition are discussed.

The closely related yeastsOgataea polymorphaandO.parapolymorphadiffer drastically from each other by sensitivity to the toxic phosphate analog vanadate. Search for genes underlying this difference revealed two genes, one designated asABV1(AlcianBlue staining,Vanadate resistance), which encodes a homologue ofSaccharomyces cerevisiaeMnn4 responsible for attachment of mannosylphosphate to glycoside chains of secretory proteins, and the other designated as itsS.cerevisiaehomologuePHO87, encoding the plasma membrane low affinity phosphate sensor/transporter. The effect of Pho87 on vanadate resistance was bidirectional, since it decreased the resistance on phosphate-depleted medium, but was required for pronounced protection against vanadate by external phosphate. This highlights the dual function of this protein as a low affinity phosphate transporter and an external phosphate sensor. Involvement of Pho87 in phosphate sensing was confirmed by its effects on regulation of the promoter of thePHO84gene, encoding a high affinity phosphate transporter. The effect of Abv1 was also complex, since it influenced Pho87 level and enhanced repression of thePHO84promoterviaa Pho87-independent pathway. Role of the identified genes in the difference in vanadate resistance betweenO.polymorphaandO.parapolymorphais discussed.

In eukaryotic cells, COPI vesicles retrieve resident proteins to the endoplasmic reticulum and mediate intra-Golgi transport. Here, we studied the Hansenula polymorpha homologue of the Saccharomyces cerevisiae RET1 gene, encoding α-COP, a subunit of the COPI protein complex. H. polymorpha ret1 mutants, which expressed truncated α-COP lacking more than 300 C-terminal amino acids, manifested an enhanced ability to secrete human urokinase-type plasminogen activator (uPA) and an inability to grow with a shortage of Ca ²⁺ ions, whereas a lack of α-COP expression was lethal. The α-COP defect also caused alteration of intracellular transport of the glycosylphosphatidylinositol-anchored protein Gas1p, secretion of abnormal uPA forms, and reductions in the levels of Pmr1p, a Golgi Ca ²⁺ -ATPase. Overexpression of Pmr1p suppressed some ret1 mutant phenotypes, namely, Ca ²⁺ dependence and enhanced uPA secretion. The role of COPI-dependent vesicular transport in cellular Ca ²⁺ homeostasis is discussed.

The use of plasmids possessing a regulatable gene coding for a site-specific recombinase together with its recognition sequences significantly facilitates genome manipulations since it allows self-excision of the portion of the genetic construct integrated into the host genome. Stable maintenance of such plasmids in Escherichia coli, which is used for plasmid preparation, requires prevention of recombinase synthesis in this host, which can be achieved by interrupting the recombinase gene with an intron. Based on this approach, Saccharomyces cerevisiae and Hansenula polymorpha self-excising vectors possessing intronated gene for Cre recombinase and its recognition sites (LoxP) were previously constructed. However, this work shows instability of the H. polymorpha vectors during plasmid maintenance in E. coli cells. This could be due to recombination between the loxP sites caused by residual expression of the cre gene. Prevention of translation reinitiation on an internal methionine codon completely solved this problem. A similar modification was made in a self-excising vector designed for S. cerevisiae. Apart from substantial improvement of yeast self-excising vectors, the obtained results also narrow down the essential part of Cre sequence.